Probiotics
The concept of probiotic microorganisms was born from the hypothesis of the Nobel Prize Elie Metchnikoff, who suggested that the consumption of bacteria able to produce fermentation (Lactobacillus) has a positive effect on the microbiota of the colon, reducing the presence of bacterial toxins and other microbial activities which have a negative impact on human health.
“Probiotics are live microorganisms that after its administration engaged in the consumer more benefits than basic nutritional natural”. Nowadays, there are a lot of references about the usefulness of probiotics to treat several disorders of gastrointestinal health, as well as studies that suggest its utility to activate the immune system and prevent allergies.
Probiotic bacteria must fulfil several requirements related to lack of toxicity, viability, adhesion and beneficial effects. The properties of each bacterial strain are unique and cannot be extrapolated to other strains of the same species (Araya, M., et al. Guidelines for the Evaluation of Probiotics in Food—Joint FAO/WHO Working Group. FAO/WHO. Editor 2002. Food and Agriculture Organization of the United Nations and World Health Organization: Ontario, Canada). Therefore, it is important to find those strains that have a better performance in all probiotic requirements.
Although the concept of probiotics was associated with intestinal microbial flora, studies conducted as early as 1988 showed that this concept could also be extended to the vaginal flora of women. A few scientific groups have been developing this idea for some years, which culminated in the identification of some strains of Lactobacillus which are useful to treat complications of vaginal health (Anukam, K. C., et al. Clinical study comparing probiotic Lactobacillus GR-1 and RC-14 with metronidazole vaginal gel to treat symptomatic bacterial vaginosis. Microbes and Infection. 2006, Vol. 8, Nos. 12-13, pages 2772-2776; Larsson, P. G., et al. Human lactobacilli as supplementation of clindamycin to patients with bacterial vaginosis reduce the recurrence rate; a 6-month, double-blind, randomized, placebo-controlled study. BMC Women's Health. 2008, Vol. 8, No. 1, page 3), thus demonstrating the concept of probiotic for vaginal health.
Healthy Vaginal Flora
The appearance of vaginal secretions and itching are the most common causes of gynaecological visits. This may be due to allergic reactions, contact with irritant agents (cloth fibber, soap, spray, etc.) or in many cases due to an infection.
The healthy vaginal flora is mostly composed of Lactobacilli, which maintain the natural acidic pH of the vagina (between 3.9 and 4.3), preventing its colonization by other microorganisms. When this natural flora is weakened, e.g. by systemic antibiotic treatments, infection by pathogenic species becomes much more likely. The most frequent complications of healthy vaginal flora are vulvovaginal candidiasis and bacterial vaginosis.
Vulvovaginal Candidiasis
Candida is a highly prevalent yeast that can be found in small numbers in the vaginal flora without any symptoms of disease. Symptoms appear when vaginal flora equilibrium is disturbed and the population of Candida increases compared to the amount of protective Lactobacilli. Typical symptoms of Candida infection include vaginal itching and burning, as well as dyspareunia and vaginism. Approximately, the 75% of the cases of the vulvovaginal candidiasis are due to Candida albicans, and an additional 15% are due to Candida glabrata (Richter, S. S., et al. Antifungal Susceptibilities of Candida Species Causing Vulvovaginitis and Epidemiology of Recurrent Cases. J. Clin. Microbiol. 2005, Vol. 43, No. 5, pages 2155-2162). It is estimated that 75% of women experience at least one episode in the course of their lifetime. Moreover, 25% of the cases are recurrent with four or more episodes per year.
Infection often occurs after treatment with antibiotic therapy prescribed with a different therapeutic aim. It is also common in women taking oral contraceptives containing estrogens, in pregnant women and in women with diabetes. Candida infections are treated with antimycotics, such as triazole drugs (e.g. fluconazole, clotrimazole, myconazole, itraconazole) or nystatin. However, it is noteworthy that strains of Candida glabrata tend to display a high resistance against such treatments, while up to 20% of the strains of Candida albicans isolated in clinics present resistance to fluconazole, one of the most typical treatments, stressing out the importance of finding new therapeutic tools for the management of vulvovaginal candidiasis.
In addition to its acidic pH, vaginal fluid contains several antimicrobial proteins, such as lysozyme, defensins and lactoferrin. During vulvovaginal candidiasis the concentration of these substances increases beyond the normal levels in approximately 25% of women (Valore, E. V., et al. Reversible Deficiency of Antimicrobial Polypeptides in Bacterial Vaginosis. Infect. Immun. 2006, Vol. 74, No. 10, pages 5693-5702). This response is usually insufficient to ward off yeasts such as Candida, as the effect of these substances is more bacteriostatic or bactericidal than antimycotic. However, since the healthy vaginal flora is mostly composed of bacteria of the Lactobacillus genus this abnormal increase in antimicrobial proteins can contribute to further delay the recuperation of the healthy flora of the vagina. Moreover, this stresses out the need of considering the resistance to these antimicrobial factors when searching for Lactobacilli suitable to be used as probiotics.
Bacterial Vaginosis
Bacterial vaginosis is caused by an overgrowth of bacterial species usually absent in the vaginal flora or found in very small amounts. The most common species are Gardnerella vaginalis and Atopobium vaginale, but a precise aetiological agent has not been found so far. About 1 out of 5 women will develop bacterial vaginosis along an interval between 6 and 12 months. Although bacterial vaginosis can cause unusual vaginal discharges and fishy odour, most cases are asymptomatic. A typical trait of bacterial vaginosis is the rise of the pH above 4.5, due to the disappearance of Lactobacilli, which further facilitates the growth of other bacterial species.
Bacterial vaginosis is treated with antibiotics, such as metronidazole and clindamicyn. However, bacterial vaginosis displays a high recurrence rate, up to 35% during the first month and up to 70% during the first year. It has also been observed that the antibiotic treatment of vaginosis can lead to vulvovaginal candidiasis as a secondary infection, since antibiotics, especially clindamicyn, also affect Lactobacilli. 
Also, it has been shown that women that have bacterial vaginosis tend to have premature babies or babies with low weight—less than 2.5 kg —. Sometimes the infection can spread to the fallopian tubes. This type of infection is called pelvic inflammatory disease (PID) and can cause infertility and damage to the fallopian tubes, increasing the risk of ectopic pregnancy. Bacterial vaginosis also increases the risk of urinary tract infections and sexually transmitted diseases.
Probiotics for the Management of Vulvovaginal Candidiasis and Bacterial Vaginosis
There are currently several probiotic strains marketed to help manage vulvovaginal candidiasis, such as Lactobacillus rhamnosus GR1, Lactobacillus fermentum RC14, Lactobacillus plantarum P17630 and Lactobacillus acidophilus NAS. However, none of them has been tested for their ability to withstand the elevated concentrations of antimicrobial factors that occur in approximately 1 out of 4 cases of infection. Plus, there is only one probiotic solution (BION Flore Intime®, composed by strains Lactobacillus rhamnosus GR1 and Lactobacillus reuteri RC14) for the management of vulvovaginal candidiasis whose efficacy claims are supported by a randomized placebo-controlled clinical trial (Martinez, R. C. R., et al. Improved treatment of vulvovaginal candidiasis with fluconazole plus probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14. Letters in Applied Microbiology. 2009, Vol. 48, No. 3, pages 269-274).
On the other hand, there are two probiotic products for the management of bacterial vaginosis whose health claims are supported by randomised placebo-controlled, clinical trials: BION Flore Intime®—Lactobacillus rhamnosus GR1 and Lactobacillus fermentum RC14—and EcoVag®—Lactobacillus gasseri DSM 14869 and Lactobacillus rhamnosus DSM 14870 (Anukam, K. C., et al. 2006, supra; Larsson, P. G., et al. 2008, supra).
The art describes other strains as potential vaginal probiotics, such as EP1436380 B1, which describes the strain Lactobacillus pentosus NCIMB 41114 isolated from a fecal culture obtained from a healthy adult individual and cultured in the presence of tetracycline. The document describes that due to its ability of suppressing the growth of Candida species and that the strain is resistant to tetracycline and related antibiotics, it can be useful to combat undesirable growth of Candida in any region of the body, in particular in Irritable Bowel Syndrome. This document is silent about the inhibitory activity of the strain against Candida glabrata, nor demonstrates the ability of the strain to adhere to the vaginal epithelium and survive in the vaginal environment or to resist to antimicrobial substances present in it, such as lysozyme.
Okkers, D. J. et al. 1999 describes that strain Lactobacillus pentosus TV35b, isolated from the posterior fornix secretions of the vagina of a prenatal patient, produced a bacteriocin-like peptide (pentocin TV35b), which is inhibitory to Candida albicans and some bacterial species. The document is silent about the inhibitory activity of the strains against Candida glabrata (Okkers, D. J. et al. Characterization of pentocin TV35b, a bacteriocin-like peptide isolated from Lactobacillus pentosus with a fungistatic effect on Candida albicans, Journal of Applied Microbiology. 1999, Vol. 87, No. 5, pages 726-734).
On the other hand, the art describes vaginal strains which do not present inhibitory activity against Candida albicans. For instance, in Dimitonova et al. 2007, the inhibitory activity of 20 strains of Lactobacilli, isolated from vaginal swabs of healthy Bulgarian women, was assessed. None of the 20 strains inhibited the growth of Candida albicans (Dimitonova, S. P. et al. Antimicrobial activity and protective properties of vaginal lactobacilli from healthy Bulgarian women. Anaerobe. 2007, Vol. 13, No. 5-6, pages 178-184).
The art also teaches that presence of inhibitory activity against Candida albicans, does not mean that the bacterial strain is also antagonist against Candida glabrata. For instance, in Pascual L. M. et al. 2008 a strain of Lactobacillus isolated from the vagina of nonpregnant, healthy, premenopausal women was identified as Lactobacillus rhamnosus L60. Lactobacillus rhamnosus L60 displayed a wide spectrum of antimicrobial activity against urogenital pathogens, and resistance to antibiotics commonly prescribed for infections caused by these pathogens. This strain was antagonist against 10 strains of Candida albicans but was not antagonist against the 3 strains of Candida glabrata tested [Pascual, L. M. et al. Lactobacillus rhamnosus L60, a potential probiotic isolated from the human vagina. Journal of General and Applied Microbiology. 2008, Vol. 54, No. 3, pages 141-148).
Therefore, the fact that the described Lactobacilli for use as potential vaginal probiotic are isolated from vagina does not mean that they present inhibitory activity against species of Candida. Moreover, the fact that a strain of Lactobacillus is antagonist against Candida albicans does not mean that also possess activity against other species of Candida, such as Candida glabrata. Thus, being antagonist for Candida species is not an inherent feature of Lactobacilli. 
WO 2012/101500 A1 describes an effervescent composition in solid form for use in vaginal applications for the treatment of vaginal infections. The effervescent composition comprises among galenic components, a probiotic bacterial strain for reducing and/or eliminating the presence of pathogenic agents in the vaginal environment. The strain is selected from a long list of the most common species used as probiotics, from Lactobacillus and Bifidobacterium genus. The document does not include a test to evaluate the properties of the strains. The invention is focused in the galenic composition designed to enhance the survival of the probiotics during their manufacture and to improve administration. The strains mentioned in the document are commercially available and the pathogens mentioned are common in vaginal pathologies.
JP 2008013502 A describes the use of a product obtained by a fermentation process of a cruciferous plant, such as e.g. broccoli, with lactic acid bacteria such as Lactobacillus pentosus, to treat or prevent Candida infections like vaginitis. Tests are done with Candida albicans deposited strains. The document is silent about the inhibitory activity of the fermented product against Candida glabrata. 
On the other hand, as explained above, antibacterial substances in vaginal fluid are increased beyond normal levels in approximately 25% of women experiencing vulvovaginal candidiasis. These substances have a significant inhibitory effect on Lactobacilli. A few Lactobacilli are currently used as vaginal probiotics; however none of them has been tested for the capacity to survive in the conditions where antimicrobial factors are increased in vaginal fluid. Moreover, none of the current vaginal probiotics has been tested for its activity specifically against Candida glabrata. C. glabrata accounts for a significant fraction of Candida infections and displays a higher resistance to current antimycotic treatments, thus presenting a greater therapeutic challenge.